CN102994486A

CN102994486A - Protease with improved stability in detergents

Info

Publication number: CN102994486A
Application number: CN2012104776578A
Authority: CN
Inventors: 吴文平; K.V.约库姆森; M.A.斯特林格
Original assignee: Novo Nordisk AS
Current assignee: Novo Nordisk AS
Priority date: 2003-10-23
Filing date: 2004-10-22
Publication date: 2013-03-27
Also published as: CN1871344A; JP2007509615A; EP1678296B1; ATE516347T1; WO2005040372A1; EP1678296A1; JP4880469B2

Abstract

The present invention relates to proteases having improved stability in detergent compositions. Further, the present invention relates to cleaning and detergent compositions comprising the proteases of the invention as well as to use of such protease in detergent compositions.

Description

In washing composition, has the proteolytic enzyme that improves stability

It is on October 22nd, 2004 that the present patent application is based on the applying date, application number is 200480031211.6 (international application no is PCT/DK2004/000730), and name is called the dividing an application of application for a patent for invention of " have improve stability proteolytic enzyme " in washing composition.

Technical field

The present invention relates to have the proteolytic enzyme that in detergent composition, improves stability.The invention still further relates to the encoding said proteins enzyme separation polynucleotide, nucleic acid construct, recombinant expression vector, comprise described nucleic acid construct host cell and for the production of with the method for using proteolytic enzyme of the present invention.In addition, the present invention relates to comprise cleaning compositions and the detergent composition of proteolytic enzyme of the present invention and relate to the purposes of described proteolytic enzyme in detergent composition.

Background of invention

In detergent industry, in the washing prescription, add application of enzymes above 30 years.The enzyme that is used for this type of prescription comprises proteolytic enzyme, lipase, amylase, cellulase, mannosidase and other enzyme or its mixture.Commercial most important enzyme is proteolytic enzyme.

In this area, for example at EP 130756 (GENENTECH) (corresponding to U.S.'s reissue patent number 34,606 (GENENCOR)); EP 214435 (HENKEL); WO 87/04461 (AMGEN); WO 87/05050 (GENEX); EP 260105 (GENENCOR); Thomas, Russell and Fersht (1985) Nature 318 375-376; Thomas, Russell and Fersht (1987) J.Mol.Biol.193803-813; Russel and Fersht Nature 328 496-500 (1987); WO 88/08028 (Genex); WO 88/08033 (Amgen); WO 95/27049 (SOLVAY S.A.); WO 95/30011 (PROCTER﹠amp; GAMBLE company); WO 95/30010 (PROCTER﹠amp; GAMBLE company); WO 95/29979 (PROCTER﹠amp; GAMBLE company); US 5.543.302 (SOLVAY S.A.); EP 251 446 (GENENCOR); WO 89/06279 (NOVOZYMES A/S); WO 91/00345 (NOVOZYMES A/S); EP 525 610 A1 (SOLVAY); WO 94/02618 (GIST-BROCADES N.V.) discloses this type of numerous ease variants.

WO 89/06270 (Novozymes A/S) discloses the detergent composition that comprises limited substrate specificity proteolytic enzyme, and wherein said proteolytic enzyme is the trypsin-like proteolytic enzyme of Methionin or arginic C-end side peptide bond of can rupturing.

And WO 94/25583 discloses the clone of coding Fusarium (Fusarium) trypsin-like proteolytic enzyme dna sequence dna and obtained the expression of active trypsin-like proteolytic enzyme from described dna sequence dna.

Yet, even if described numerous useful proteolytic enzyme and ease variants, still still need further to improve proteolytic enzyme or ease variants for numerous industrial uses.

Particularly, because the material that exists in the washing composition tends to reduce the albumen Enzymic stability, so the stability problem of proteolytic enzyme in washing composition become especially obvious.

Therefore, target of the present invention provides the proteolytic enzyme of improvement, and described proteolytic enzyme is applicable in the washing composition of laundry for example and/or hard surface cleaning.

The invention summary

The present invention is relating to the proteolytic enzyme with improved stability in washing composition aspect first, described proteolytic enzyme is selected from

A. has the proteolytic enzyme with the aminoacid sequence of aminoacid sequence at least 73% identity shown in the 1-266 amino acids of SEQ ID NO:2;

B. by proteolytic enzyme coded with the nucleotide sequence of following sequence hybridization under low stringency condition:

(i) complementary strand of the nucleotide sequence shown in the 127-804 position Nucleotide of SEQ ID NO:1, or

(ii) subsequence of (i) of at least 100 Nucleotide; And

C. compare with aminoacid sequence shown in the 1-266 amino acids of SEQ ID NO:2 have 1-50, preferred 1-40 or 1-30, more preferably 1-20, the proteolytic enzyme of 1-10 amino acid replacement most preferably.

The present invention relates to the separation polynucleotide of the nucleotide sequence that comprises code book invention proteolytic enzyme aspect second.

The present invention relates to the separation polynucleotide of proteins encoded enzyme aspect the 3rd, and described polynucleotide are selected from:

A. has the nucleotide sequence with nucleotide sequence at least 80% identity shown in the 52-804 position Nucleotide of SEQ ID NO:1; With

B. under low stringency condition with the nucleotide sequence of following sequence hybridization:

(i) complementary strand of nucleotide sequence shown in the 52-804 position Nucleotide of SEQ ID NO:1; Or

(ii) subsequence of (i) of at least 100 Nucleotide.

The present invention relates to the nucleic acid construct that comprises nucleotide sequence of the present invention aspect the 4th, wherein said nucleotide sequence effectively is connected with one or more control sequences that can instruct proteolytic enzyme to express in suitable host.

The present invention relates to the recombinant expression vector that comprises nucleic acid construct of the present invention, promotor and transcription termination signal and translation termination signal aspect the 5th.

The present invention relates to the recombinant host cell that comprises nucleic acid construct of the present invention aspect the 6th.

The present invention relates to the method for production proteolytic enzyme of the present invention aspect the 7th, and described method comprises:

(a) inducing cultivation recombinant host cell of the present invention under the condition that produces described proteolytic enzyme; With

(b) reclaim proteolytic enzyme.

The present invention relates to cleaning compositions or detergent composition, the preferred laundry composition that comprises proteolytic enzyme of the present invention or washes the dish composition aspect the 8th.

Other aspects of the present invention relate to the purposes of proteolytic enzyme of the present invention in cleaning compositions or detergent composition; The method that is used for cleaning or washing hard surface or clothing, described method comprise hard surface or clothing are contacted with the present composition.

Definition

Before more discussing the present invention in detail, at first be defined as follows term and convention.

Amino acid whose name

The A=Ala=L-Ala

The V=Val=α-amino-isovaleric acid

The L=Leu=leucine

The I=Ile=Isoleucine

The P=Pro=proline(Pro)

The F=Phe=phenylalanine

The W=Trp=tryptophane

The M=Met=methionine(Met)

The G=Gly=glycine

The S=Ser=Serine

The T=Thr=Threonine

The C=Cys=halfcystine

Y=Tyr=tyrosine

The N=Asn=l-asparagine

The Q=Gln=glutamine

The D=Asp=aspartic acid

E=Glu=L-glutamic acid

K=Lys=Methionin

The R=Arg=arginine

The H=His=Histidine

The X=Xaa=arbitrary amino acid

The name of nucleic acid

The A=VITAMIN B4

The G=guanine

The C=cytosine(Cyt)

T=thymus pyrimidine (only in DNA)

U=uridylic (only in RNA)

Proteolytic enzyme

The enzyme of amido linkage in the fracture protein substrate is classified as proteolytic enzyme or (being interchangeably) peptase (seeing Walsh, 1979, Enzymatic Reaction Mechanisms.W.H.Freeman and Company, San Francisco, the 3rd chapter).

Serine protease

Serine protease is catalysis peptide bond hydrolysis and the enzyme (White that has an essential serine residue in the avtive spot of enzyme, Handler and Smith, 1973, " Principles of Biochemistry ", the 5th edition, McGraw-Hill Book Company, NY, 271-272 page or leaf).

The molecular weight ranges of bacterial serine proteolytic enzyme is 20,000-45,000 dalton.They can be suppressed by diisopropylfluorophosphate.The simple terminal ester of bacterial serine protease hydrolysis and its activity are similar to the eucaryon Quimotrase that is similarly serine protease.More the term Sumizyme MP of narrow sense then contains and covers a subclass, and it reflects that some serine protease has high best pH (9.0-11.0) (summary is seen Priest (1977) Bacteriological Rev.41 711-753).

Trypsin-like proteolytic enzyme

Term " trypsin-like proteolytic enzyme " is intended to refer to have the proteolytic enzyme that is similar to tryptic activity in the present context, namely can rupture to be positioned at the enzyme of Methionin or arginic C-terminal side peptide bond.As described in EXAMPLE IV in following materials and methods part, measure the trypsin-like protease activity based on the assay method of trypsinase substrate fracture.

Parent proteolytic enzyme

Parent proteolytic enzyme can be the proteolytic enzyme that separates from natural source, wherein can carry out follow-up modification to this proteolytic enzyme when keeping the proteolytic enzyme feature.And parent proteolytic enzyme can also be by such as J.E.Ness etc., Nature Biotechnology, the proteolytic enzyme of the described DNA shuffling technology preparation of 17,893-896 (1999).Alternatively, term " parent proteolytic enzyme " can be called " wild-type protease ".

The albumen modification of enzyme

Term used herein " modification " is defined as the chemical that comprises proteolytic enzyme is modified and the genetic manipulation of the DNA of proteins encoded enzyme.Described modification can be that amino acid side chain is replaced, substituting, lacking and/or inserting at purpose amino acid place.

Ease variants

In the context of the present invention, term protease variant or the proteolytic enzyme that has suddenlyd change mean a kind of biogenic proteolytic enzyme by expressing mutator gene, wherein said mutator gene is derived from the parent microorganism that has original gene or maternal gene and produce corresponding parent enzyme, and producing this protease mutant in order can to express in the host time becomes mutator gene with the maternal gene sudden change.Similarly, mutator gene also can be derived from the maternal gene that produces through the DNA shuffling technology.

The homologous protein enzyme sequence

In the present context, the homology between the two seed amino acid sequences is described by parameter " identity ".

In order to determine the identity degree between two kinds of proteolytic enzyme, can use the GAP program that the AlignX of the Vector NTI routine package v8 of default setting uses.Result from program also calculates " identity percentage ratio " between two kinds of sequences except the amino acid comparison.

Based on this description, those skilled in the art can conventionally identify modifiable suitable homologous protein enzyme and corresponding homology avtive spot ring zone according to the present invention.

Separate polynucleotide

As used herein, term " separation polynucleotide " refers to separate with purifying and therefore is in the polynucleotide that are applicable to genetically engineered protein production system form.This type of isolated molecule can be those molecules that separate from its natural surroundings, and comprises cDNA clone and genomic clone and be derived from the polynucleotide that site-directed spontaneous generation experiment was tested or was derived from DNA reorganization.Separation polynucleotide of the present invention do not contain usually with it other gene of combination, but can comprise 5 ' and 3 ' non-translational region, such as promotor and terminator.The zone of identifying institute's combination is easy to do (seeing for example Dynan and Tijan, Nature 316:774-78,1985) for the ordinary skill in the art.Term " separated nucleic acid sequence " can alternatively be called " DNA isolation sequence ", " cloning nucleic acid sequences " or " cloned dna sequence ".

Isolated protein

When showing this protein when being applied to protein, term " separation " broken away from its natural surroundings.

In a preferred mode, isolated protein does not contain other oroteins substantially, especially other homologous protein (i.e. " homology impurity " (seeing lower)).

As determining by SDS-PAGE, isolated protein purity greater than 10%, be preferably greater than 20%, more preferably greater than 30%.In addition, preferably provide such as the protein by the determined highly purified form of SDS-PAGE, namely purity is greater than 40%, greater than 60%, greater than 80%, more preferably greater than 95% and most preferably greater than 99%.

Term " isolated protein " can alternatively be called " protein purification ".

Homology impurity

Term " homology impurity " means to be derived from any impurity (for example other polypeptide except proteolytic enzyme of the present invention) in the homologous cell of initial acquisition proteolytic enzyme of the present invention.

Obtain certainly

As used herein polynucleotide and/or the proteolytic enzyme of the term relevant with specified microorganisms source " obtain from " cell generation that mean to be produced by particular source or that inserted by the source gene.

Substrate

The term " substrate " relevant with the substrate of proteolytic enzyme should be interpreted as the generalized form of this term as used herein, as comprises the compound that comprises at least a peptide bond to the protease hydrolysis sensitivity.

Product

Should be interpreted as comprising in the context of the present invention the product of the hydrolysis reaction that relates to proteolytic enzyme such as the employed term " product " relevant with the enzyme reaction product that is derived from proteolytic enzyme.Product can be the substrate in the follow-up hydrolysis reaction.

Scourability

In the present context, term " scourability " is removed stain, is particularly removed the ability of the egg mark on the object as the expression enzyme in for example washing or hard surface cleaning process.Also can be referring to " performance test of pattern detergent washing " in the example VII A.

The accompanying drawing summary

Fig. 1 has described albumen enzymeinhibition spectrogram of the present invention.

Fig. 2 has described the substrate specificity of proteolytic enzyme of the present invention.

Fig. 3 has described the temperature spectrogram of proteolytic enzyme of the present invention.

Fig. 4 has described the pH spectrogram of the proteolytic enzyme of the present invention of measuring such as the DMC assay method.

Fig. 5 has described the pH spectrogram of the proteolytic enzyme of the present invention of measuring such as ACZL-casein-assay method.

Fig. 6 has described the pH-stability spectrogram of proteolytic enzyme of the present invention.

Detailed Description Of The Invention

In the present invention first cause concern aspect, have the trypsin-like proteolytic enzyme that improves stability and be the protein isolate enzyme that has at least 73% identity with aminoacid sequence shown in the 1-266 amino acids of SEQ ID NO:2 (being ripe proteolytic enzyme).

In an embodiment that causes concern, proteolytic enzyme of the present invention and aminoacid sequence shown in the 1-266 amino acids of SEQ ID NO:2 have greater than 75% or greater than 80% or greater than 85% or greater than 90% or greater than 92% or greater than 94% or greater than 96% or greater than 97% or greater than 98% or greater than 99% identity.

Another cause concern aspect, described proteolytic enzyme is the ease variants with aminoacid sequence shown in the-25-266 amino acids of SEQ ID NO:2, described ease variants comprises the substituting of one or more amino-acid residues, disappearance and/or inserts.

The calculating of sequence alignment and identity value can use to protein and DNA comparison all useful complete Smith-Waterman comparison carry out.Aminoacid sequence can use the AlignX application program comparison of the Vector NTI routine package v8 of default setting, this application program is used and is improved ClustalW algorithm (Thompson, J.D., Higgins, D.G. with Gibson T.J., 1994), blosum62mt2 score matrix, opening point penalty 10 and breach extend point penalty 0.1.

By to the aminoacid sequence of proteolytic enzyme with SEQ ID NO:2 aminoacid sequence and think that prior art compares near carrying out this kind between the aminoacid sequence of proteolytic enzyme, found that identity is 73% mature protein.

Another causes in the embodiment of concern in the present invention, the protein isolate enzyme is by such nucleic acid sequence encoding, wherein said nucleotide sequence is under low stringency condition, preferably under medium stringent condition, more preferably under high stringent condition with (i) complementary strand of nucleotide sequence shown in the 127-804 position Nucleotide of SEQ ID NO:1, or (ii) subsequence hybridization (J.Sambrook of (i) of at least 100 Nucleotide, E.F.Fritsch and T.Maniatus, 1989, Molecular Cloning, A Laboratory Manual, second edition, ColdSpring Harbor, New York).

The subsequence of nucleic acid array complementation chain can be at least 100 Nucleotide or preferably at least 200 Nucleotide or at least 300 Nucleotide shown in the 127-804 position Nucleotide of SEQ ID NO:1, or at least 400 Nucleotide.And, the proteolytic enzyme fragment that subsequence should be encoded and be had proteolytic activity.Proteolytic enzyme can also be proteolytic enzyme allele variant or the fragment with proteolytic activity.

Aminoacid sequence or its fragment of the nucleotide sequence of SEQ ID NO:1 or its subsequence and SEQ ID NO:2 can be used for the designing nucleic acid probe, in order to identify in the strain that never belongs to together or plant according to method well-known in the art and clones coding has the DNA of the subtilase of proteolytic activity.Particularly, this type of probe is used in the standard DNA trace method genomic dna that belongs to purpose or plant or cDNA hybridization in order to identify and separate its corresponding gene.This type of probe is obvious shorter than complete sequence, but length should at least 15, preferred 25 and more preferably 35 Nucleotide.Also can use longer probe.Dna probe and rna probe all can use.Probe generally is (for example using for use in detecting corresponding gene of mark ³²P, ³H, ³⁵S, vitamin H or avidin 9 white marker).This type of probe is included among the present invention.

Therefore, the genome dna library for preparing from other this type of biology or cDNA library can screen the DNA with above-mentioned probe hybridization and code book invention subtilase.Can separate by the agarose known to the those of skill in the art or polyacrylamide gel electrophoresis or other isolation technique from other this type of biological genomic dna or other DNA.DNA or DNA isolation from the library can shift and be fixed on nitrocellulose or other appropriate carrier material.In order to identify and clone or the DNA of SEQ ID NO:1 or its subsequence homology, described solid support material is used for southern blotting technique.For the purposes of the present invention, hybridization refers to nucleotide sequence and hybridizes under the paramount stringent condition of low stringency condition corresponding to the labeling nucleic acid probe of nucleotide sequence shown in the SEQ ID NO:1, its complementary strand or its subsequence.Use X-ray film to detect under this type of condition molecule with nucleic acid probe hybridization.

For the long probe of at least 100 length of nucleotides, with the paramount stringent condition of low stringency condition be defined as after standard DNA trace method in 42 ℃ 5 * SSPE, 0.3% SDS, 200 μ g/ml sheared and sex change salmon sperm DNA and 25% methane amide (for low stringency condition), 35% methane amide (for medium stringent condition) or 50% methane amide (for high stringent condition) in prehybridization and hybridization.

Long probe at least 100 length of nucleotides, solid support material with 2 * SSC, 0.2% SDS in preferably at least 50 ℃ (low stringency conditions), more preferably at least 55 ℃ (medium stringent conditions) even be more preferably 65 ℃ (high stringent conditions) lower final washing three times, each 15 minutes.

For the short probe of about 15 Nucleotide of length to about 70 Nucleotide, stringent condition is defined as after standard DNA trace method in being lower than according to Bolton and McCarthy (1962, Proceedings of the National Academy of Sciences USA 48:1390) method of calculation are calculated under the temperature of 5-10 ℃ of Tm value, at 0.9M NaCl, 0.09M Tris-HCl pH 7.6,6mMEDTA, 0.5%NP-40,1 * Denhardt solution, the 1mM trisodium phosphate, the 1mM SODIUM PHOSPHATE, MONOBASIC, 0.1mMATP and carry out prehybridization in the 0.2mg/ml yeast rna, hybridization and post-hybridization washing.

For the short probe of about 15 Nucleotide of length to about 70 Nucleotide, solid support material calculates in being lower than under the temperature of 5-10 ℃ of Tm value, washing once (15 minutes) and with 6 * SCC washed twice (each 15 minutes) in adding 6 * SCC of 0.1%SDS.

This area is well-known, and conservative replacement of what is called that a kind of amino-acid residue replaces to similar amino-acid residue only produces slight the change on the feature that is expected at enzyme.

Following table 1 has been listed the group that conserved amino acid is replaced.

Table 1

Conservative amino acid replacement

Therefore, in another embodiment that attracts people's attention, the proteolytic enzyme with SEQ ID NO:2 aminoacid sequence has substituting, lack and/or inserting of one or more amino-acid residues simultaneously in the present invention.

And the institute's protein isolate enzyme (preferred purified form) that has immunochemistry identity or partial immunity chemistry identity with the proteolytic enzyme with SEQ ID NO:2 aminoacid sequence also is in the scope of the invention.Immuno-chemical property can be detected by immunological cross-reaction identity by the two-way immunodiffusion(ID) method of well-known Ouchterlony to be determined.Particularly, according to Harboe and Ingild at N.H.Axelsen, J.

The A Manual of Quantitative Immunoelectrophoresis that edits with B.Weeks, BlackwellScientific Publications, 1973, the Immuno-chemistry in Practice of the 23rd chapter or Johnstone and Thorpe, Blackwell Scientific Publications, 1982 (more specifically at the 27-31 page or leaf) described method immunize rabbits (or other rodent) preparation antiserum(antisera), wherein said antiserum(antisera) comprises and the proteolytic enzyme epi-position generation immune response with SEQ ID NO:2 aminoacid sequence or the polyclonal antibody of combination.Proteolytic enzyme with immunochemistry identity is the proteolytic enzyme that reacts in the same manner with antiserum(antisera), described same way as for example when using the specific immune chemical technology precipitation merge fully, identical precipitation morphology and/or identical electrophoretic mobility.Axelsen, Bock and

At N.H.Axelsen, J.

With the A Manual of Quantitativeimmunoelectrophoresis that B.Weeks edits, Blackwell Scientific Publications has deeply explained immunochemistry identity in 1973, the 10 chapters.Proteolytic enzyme with partial immunity chemistry identity is and the proteolytic enzyme of antiserum(antisera) with the reaction of part same way as, identical precipitation morphology and/or the identical electrophoretic mobility of part of meromixis, part that described part same way as for example precipitates when using the specific immune chemical technology.Bock and Axelsen be at N.H.Axelsen, J. With the A Manual ofQuantitative immunoelectrophoresis that B.Weeks edits, Blackwell Scientific Publications, 1973, partial immunity chemistry identity has been described in the Chapter 11.

Antibody can also be monoclonal antibody.Monoclonal antibody can be according to such as E.Harlow and D.Lane, editor, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press, ColdSpring Harbor, the method preparation of New York and using.

The inventor has separated coding to have the gene of the subtilase of aminoacid sequence as shown in SEQ ID NO:2 and is inserted into to intestinal bacteria (Escherichia coli) NN049696.The microbial strains preservation budapest treaty that is used for patented procedure according to international recognition, the intestinal bacteria NN049696 strain of carrying described gene is preserved in microbial preservation center (the MascheroderWeg 1B of Germany on February 8th, 2000, D-38124 Braunschweig, Germany) and to specify preserving number be DSM 15940.

In embodiment that attracts people's attention of the present invention, described proteolytic enzyme with had by the coded proteolytic enzyme of protease-encoding part of polynucleotide greater than 73.0% or greater than 75.0% or greater than 80.0% or greater than 85.0% or greater than 90.0% or greater than 92.0% or greater than 94.0% or greater than 96.0% or greater than 97.0% or greater than 98.0% or greater than 99.0% identity, the protease-encoding part of wherein said polynucleotide has been cloned and has entered plasmid fragment among the preserving number DSM 15940 intestinal bacteria NN049696.

As mentioned above, proteolytic enzyme of the present invention shows the stability of improvement in washing composition.Therefore, can select effective and preferred proteolytic enzyme in order to make those of skill in the art, the inventor provides and can have been implemented easily so that the suitable method of testing of the assessment proteolytic enzyme performance of discussing by those of skill in the art for this purpose.

Therefore, can use herein in the EXAMPLE V disclosed stability test to assess selected albumen Enzymic stability.That is to say, when proteolytic enzyme is mixed in the standard wash agent composition, the albumen Enzymic stability that can use this embodiment assessment to compare with reference system (being mixed in the model identical detergent system and under the same conditions test).

One of the present invention attract people's attention aspect in, has at least 40% residual activity when described proteolytic enzyme is at 30 ℃ when test in " stability in the EXAMPLE V washing composition ", in the time of 30 ℃, has at least 50% residual activity, for example in the time of 30 ℃, have at least 60% residual activity, more preferably in the time of 30 ℃, have at least 70% residual activity.

In aspect another attracts people's attention in the present invention, has at least 40% residual activity when described proteolytic enzyme is at 35 ℃ when test in " stability in the EXAMPLE V washing composition ", in the time of 35 ℃, has at least 50% residual activity, for example in the time of 35 ℃, have at least 60% residual activity, more preferably in the time of 35 ℃, have at least 70% residual activity.

Therefore, the proteolytic enzyme that attracts people's attention especially for the clothes washing purpose is such proteolytic enzyme, when this proteolytic enzyme as show the stability of comparing improvement with the reference enzyme of testing under the same terms herein in " steady testing " described pattern detergent composition that comprises following ingredients during test:

Proteolytic enzyme of the present invention can be by being used for the artificial multifarious standard technique structure that produces, (see WO 95/22625 and J.E.Ness etc. such as the DNA reorganization by different proteinase genes, NatureBiotechnology, 17,893-896 (1999)).

Obviously, proteolytic enzyme of the present invention also can separate from natural origin, be that proteolytic enzyme of the present invention can be fungi polypeptide for example, and be more preferably filamentous fungus proteolytic enzyme, such as Fusarium, the mould genus of top spore (Acremonium), Aspergillus (Aspergillus), aureobasidium genus (Aureobasidium), Cryptococcus (Cryptococcus), Filibasidium, Humicola (Humicola), Magnaporthe, Mucor (Mucor), myceliophthora (Myceliophthora), Neocallimastix, Neurospora (Neurospora), paecilomyces (Paecilomyces), Penicillium (Penicillium), Piromyces, Schizophyllum (Schizophyllum), Talaromyces (Talaromyces), thermophilic ascomycete belongs to (Thermoascus), Thielavia (Thielavia), the proteolytic enzyme of Tolypocladium (Tolypocladium) or Trichoderma (Trichoderma); Or endotrypsin for example mycocandida (Candida), genus kluyveromyces (Kluyveromyces), Pichia (Pichia), yeast belong (Saccharomyces), Schizosaccharomyces (Schizosaccharomyces) or inferior sieve yeast belong (Yarrowia) proteolytic enzyme.

In the embodiment that another attracts people's attention, proteolytic enzyme is bar spore shape sickle spore (Fusariumbactridioides), Fusarium cerealis, Fusarium crookwellense, machete sickle spore (Fusariumculmorum), fusarium graminaria (Fusarium graminearum), the red sickle spore of standing grain (Fusariumgraminum), different spore sickle spore (Fusarium heterosporum), albizzia sickle spore (Fusarium negundi), point sickle spore (Fusarium oxysporum), racemosus sickle spore (Fusarium reticulatum), pink sickle spore (Fusarium roseum), Williams Elder Twig sickle spore (Fusarium sambucinum), colour of skin sickle spore (Fusariumsarcochroum), intend branch spore sickle spore (Fusarium sporotrichioides), sulphur look Fusariumsp (Fusariumsulphureum), Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, microorganism Aspergillus aculeatus (Aspergillus aculeatus), Aspergillus awamori (Aspergillus awamori), smelly aspergillus (Aspergillus foetidus), aspergillus japonicus (Aspergillus japonicus), Aspergillus nidulans (Aspergillus nidulans), aspergillus niger (Aspergillus niger), aspergillus oryzae (Aspergillus oryzae), lonely humicola lanuginosa (Humicola insolens), pubescence humicola lanuginosa (Humicola lanuginose), rice black wool mould (Mucor miehei), Myceliphthora thermophila, Neuraspora crassa (Neurospora crassa), penicillium purpurogenum (Penicillium pur-purogenum), trichoderma harziarum (Trichoderma harzianum), healthy and free from worry wood mould (Trichoderma koningii), long handle wood mould (Trichoderma longibrachiatum), Li Shi wood mould (Trichoderma reesei) or viride (Trichoderma viride) proteolytic enzyme.

In an embodiment that attracts people's attention, proteolytic enzyme is saccharomyces carlsbergensis (Saccharomycescarlsbergensis), yeast saccharomyces cerevisiae (Saccharomyces cerevisiae), saccharomyces diastaticus (Saccharomycesdiastaticus), Saccharomyces douglasii, Crewe not yeast (Saccharomyces kluyveri), promise ground yeast (Saccharomyces norbensis) or Saccharomyces oviformis proteolytic enzyme.

Should be appreciated that for above-mentioned species to the present invention includes coordinator such as anamorph on good working condition and imperfect state and other taxonomy, and no matter whether its kind name is known.Those skilled in the art is the identity of the suitable coordinator of identification easily.

Proteolytic enzyme of the present invention can also be bacteria protease, such as proteolytic enzyme such as bacillus (Bacillus) polypeptide of gram positive bacterium, for example Alkaliphilic bacillus (Bacillus alkalophilus), bacillus amyloliquefaciens (Bacillus amyloliquefaciens), bacillus brevis (Bacillus brevis), Bacillus circulans (Bacillus circulans), Bacillus coagulans (Bacillus coagulans), Bacillus lautus, bacillus lentus (Bacillus lentus), Bacillus licheniformis (Bacillus licheniformis), bacillus megaterium (Bacillus megaterium), bacstearothermophilus (Bacillus stearothermophilus), subtilis (Bacillus subtilis) or bacillus thuringiensis (Bacillus thuringiensis) proteolytic enzyme; Or streptomyces (Streptomyces) proteolytic enzyme such as muta lead mycillin (Streptomyces lividans) or mouse ash streptomycete (Streptomyces murinus) proteolytic enzyme; Perhaps gram negative bacterium proteolytic enzyme such as intestinal bacteria or Rhodopseudomonas (Pseudomonas) proteolytic enzyme.

For example American type culture collection (ATCC), Germany microbial preservation center (DSM), Dutch fungi strain preservation center (CBS) and U.S. north Agricultural Research Institute culture collection center (NRRL) obtain the strain of these species to the public from numerous DSMZs easily.

And, can use above-mentioned probe to originate from other, comprise and identify and obtain this type of proteolytic enzyme in the microorganism that from nature (for example soil, compost, water etc.), separates.Being used for from the technology of natural surroundings separate microorganism is that this area is well-known.Similarly, the genomic library or the cDNA library that screen another kind of microorganism also can obtain polynucleotide.In case use probe in detecting to the polynucleotide of proteins encoded enzyme, then the technical point known to available those of ordinary skills from or clone this sequence (see such as Sambrook etc., 1989, on seeing).

Usually, can use for clone gene with to described gene and import standard method that (at random and/or site-directed) insert in order to obtain proteolytic enzyme of the present invention.To further describing of appropriate technology, can be with reference to herein embodiment (videing infra) and Sambrook etc., (1989) Molecular cloning:A laboratorymanual, Cold Spring Harbor lab., Cold Spring Harbor, NY; Ausubel, F.M. etc. (editor) " Current protocols in Molecular Biology " John Wiley and Sons, 1995; Harwood, C.R. and Cutting, S.M. (editor) " Molecular Biological Methods forBacillus ", John Wiley and Sons, 1990; And WO 96/34946.

In addition, proteolytic enzyme of the present invention can manually be created multifarious standard technique and make up by being used for, and for example (WO 95/22625 for the DNA reorganization by different proteinase genes; Stemmer WPC, Nature370:389-91 (1994)).

Polynucleotide

The invention still further relates to the separation polynucleotide of code book invention proteolytic enzyme.

In an embodiment that attracts people's attention, the identity of polynucleotide and the polynucleotide shown in the 52-804 position Nucleotide of SEQ ID NO:1 is at least 86%, such as at least 87%, for example at least 88%, preferably at least 89%, such as at least 90%, for example at least 91%, more preferably at least 92%, such as at least 93%, for example at least 94%, most preferably at least 95%, such as at least 96%, for example at least 97%, particularly at least 98%, preferably at least 99%.In the present invention in another embodiment that attracts people's attention, polynucleotide comprise the fragment that shown in the 127-804 position Nucleotide of SEQ ID NO:1 polynucleotide, its allele variant or its can code book invention proteolytic enzyme.Obviously, polynucleotide can be comprised of the polynucleotide shown in the 127-804 position Nucleotide of SEQID NO:1.

The present invention also comprises the polynucleotide that coding has the polypeptide of SEQ ID NO:2 aminoacid sequence, and wherein said polynucleotide are different from SEQ ID NO:2 because of codon degeneracy.The subsequence of the SEQ ID NO:1 of the SEQ ID NO:2 fragment that the invention still further relates to encodes has proteolytic activity.

The subsequence of SEQ ID NO:1 is the polynucleotide that comprise the 52-804 position Nucleotide of SEQ ID NO:1, has just removed 5 ' terminal and/or 3 ' terminal one or more Nucleotide.

For separating of or the technology of the polynucleotide of clones coding polypeptide be this area crowd know and comprise from genomic dna separate, preparation or its combination from cDNA.The expression library antibody screening method that can use for example well-known polymerase chain reaction (PCR) or be intended to detect the institute's cloned DNA fragment with common structure feature realizes clone's polynucleotide of the present invention from this genome.See such as Innis etc., 1990, PCR:A Guide to Methods and Application, Academic Press, New York.Can use other nucleic acid amplification method, for example ligase chain reaction (LCR) (LCR), connection activated transcription method (LAT) and acid sequence dependent amplification method (NASBA).

Separate polynucleotide and can polynucleotide be obtained from the standard cloning process that its natural place migrates to its different loci that can regenerate by being intended to of using in the genetically engineered for example.Described cloning process comprises to be sheared with the purpose nucleic acid fragment that separates the polynucleotide that contain the proteins encoded enzyme, is integrated into host cell with fragment insertion vector molecule and with recombinant vectors, will copy a plurality of copies or the clone of polynucleotide in described host cell.Described polynucleotide can be originated for genome, cDNA source, RNA source, semi-synthetic source, synthetic source or its combination.

For the purposes of the present invention, such as the identity degree between two kinds of polynucleotide of above-mentioned mensuration.

To the polynucleotide of code book invention proteolytic enzyme modify for the basic similarly albumen Enzyme Production of proteolytic enzyme may be essential.The non-natural existence form of term and proteolytic enzyme " substantially similar " finger protein enzyme.These proteolytic enzyme are different from the proteolytic enzyme that separates from its natural origin, different variants such as specific activity, thermostability, best pH in some through engineering approaches mode.Polynucleotide (for example its subsequence) that the variant sequence can partly exist based on the peptide coding as SEQ ID NO:1 and making up, and/or select the nucleotide substitution of (for the production of enzyme) or make up by importing the nucleotide substitution that can produce the different aminoacids sequence by importing can not produce the another kind of aminoacid sequence of the coded proteolytic enzyme of nucleotide sequence but meet the host living beings codon.See such as Ford etc., 1991, Protein Expression and Purification 2:95-107 for the general introduction of nucleotide substitution.

Those skilled in the art know, and this type of substitutes and can carry out and still produce active protease beyond the function critical area of molecule.Can be according to means known in the art, (see for example Cunningham and Wells such as site directed mutation or Alanine-scanning sudden change, 1989, Science 244:1081-1085) identify for the activity of the polypeptide of polynucleotide encoding that the present invention separates essential and the therefore preferred amino-acid residue that does not substitute.In the Alanine-scanning mutating technology, each positive charge residue place introduces sudden change in molecule, and the proteolytic activity of test gained mutating molecule is to identify the vital amino-acid residue of molecular activity.The site of substrate-enzyme interacting also can determine by three-dimensional structural analysis, and described three-dimensional structural analysis can be by (seeing such as deVos etc., 1992, Science 255:306-312 such as nuclear magnetic resonance spectroscopy, crystallography or photoaffinity labeling technical measurement; Smith etc., 1992, Journal ofMolecular Biology224:899-904; Wlodaver etc., 1992, FEBS Letters 309:59-64).

Nucleic acid construct

The invention still further relates to the nucleic acid construct that comprises polynucleotide of the present invention, wherein said polynucleotide effectively are connected with one or more control sequences that can instruct polypeptide to express in suitable host cell.

In order to make proteolytic enzyme expression, in many ways the separation polynucleotide of operate coding proteolytic enzyme of the present invention.It may be that expect or essential before insertion vector polynucleotide being operated, and this depends on expression vector.The technology of utilizing recombinant DNA method to modify polynucleotide is well-known in the art.

Control sequence comprise to express proteolytic enzyme of the present invention institute must or favourable all the components.For the polynucleotide of proteins encoded enzyme, each control sequence can be natural or external.This type of control sequence includes but not limited to leader sequence, polyadenylation sequence, propeptide sequence, promotor, signal peptide sequence and transcription terminator.Control sequence comprises promotor and transcription termination signal and translation termination signal at least.The control sequence that contains joint can be provided, and wherein said joint is used for introducing is convenient to the special restriction enzyme site that control sequence is connected with the coding region of the polynucleotide of proteins encoded enzyme.

Control sequence can be suitable promoter sequence, the sequence that namely can be identified by host cell when nucleotide sequence is expressed.Promoter sequence comprises the transcriptional control sequence of mediating proteins expression of enzymes.Promotor can be any polynucleotide of performance transcriptional activity in selected host cell, it comprises the mutant promotor, blocks promotor and hybrid promoter, and can obtain from coding and the extracellular protease of host cell homology or allos or the gene of intracellular protease.

Being used for instructing nucleic acid construct of the present invention is from aspergillus oryzae TAKA amylase gene at the example of the suitable promotor of filamentous fungal host cell transcription, rhizomucor miehei (Rhizomucor miehei) aspartate protease gene, the neutral α-amylase gene of aspergillus niger, aspergillus niger acid acceptance α-amylase gene, aspergillus niger or Aspergillus awamori glucoamylase (glaA) gene, the rhizomucor miehei lipase gene, the aspergillus oryzae alkaline protease gene, aspergillus oryzae triose-phosphate isomerase gene, the promotor (WO 96/00787) that Aspergillus nidulans acetamidase gene and sharp sickle spore trypsin-like proteinase gene obtain and NA2-tpi promotor (from the heterozygote of the promotor of the neutral α-amylase gene of aspergillus niger and aspergillus oryzae triose-phosphate isomerase gene) with and the sudden change promotor, block promotor and hybrid promoter.

Useful promotor is the promotor that obtains from yeast saccharomyces cerevisiae Hydratase, phosphoenolpyruvate (ENO-1) gene, yeast saccharomyces cerevisiae galactokinase (GAL1) gene, Ethanol in Saccharomyces cerevisiae desaturase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP) gene and yeast saccharomyces cerevisiae 3-phoshoglyceric acid kinase gene in yeast host.Other promotor that is used for yeast host cell is described in Romanos etc., 1992, Yeast 8:423-488.

Be used for instructing nucleic acid construct of the present invention to transcribe, especially the example in the suitable promotor of bacterial host cell transcription is from intestinal bacteria lac operator gene, streptomyces coelicolor (Streptomyces coelicolor) gelase gene (dagA), subtilis type froctosan saccharase gene (sacB), bacillus licheniformis alpha amylase gene (amyL), bacstearothermophilus maltogenic amylase gene (amyM), bacillus amyloliquefaciens α-amylase gene (amyQ), Bacillus licheniformis penicillinase gene (penP), β-lactamase gene (the Villa-Kamaroff etc. of subtilis xylA and xylB gene and protokaryon, 1978, Proceedings of theNational Academy of Sciences USA 75:3727-3731) promotor and the tac promotor (DeBoer etc. that obtain, 1983, Proceedings of the National Academy of Sciences USA 80:21-25).Other promotor be described in " Useful proteins from recombinant bacteria " (ScientificAmerican, 1980,242:74-94) and Sambrook etc., 1989, the same.

Control sequence can also be suitable transcription termination sequence, and namely host cell is identified the sequence that stops transcribing.Terminator sequence effectively is connected with 3 ' end of the polynucleotide of proteins encoded enzyme.Any terminator that has function in selected host cell can be used for the present invention.

The preferred terminator that is used for filamentous fungal host cell is the terminator that obtains from aspergillus oryzae TAKA amylase gene, aspergillus niger glucoamylase gene, Aspergillus nidulans anthranilic acid synthase gene, aspergillus niger α Polyglucosidase gene and sharp sickle spore trypsin-like proteinase gene.

The preferred terminator that is used for yeast host cell is the terminator that obtains from yeast saccharomyces cerevisiae enolase gene, brewing yeast cell pigment C (CYC1) gene and yeast saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase gene.Other terminator that is used for yeast host cell is described in Romanos etc., and 1992, the same.

Control sequence can also be suitable leader sequence, the mRNA untranslated zone of namely the host cell translation being overstated and wanting.Leader sequence effectively is connected in the polynucleotide 5 ' end of coded polypeptide.Any leader sequence that has function in selected host cell can be used for the present invention.

The preferred leader sequence that is used for filamentous fungal host cell obtains from aspergillus oryzae TAKA amylase gene and Aspergillus nidulans triose-phosphate isomerase gene.

The suitable leader sequence that is used for yeast host cell obtains from yeast saccharomyces cerevisiae Hydratase, phosphoenolpyruvate (ENO-1) gene, yeast saccharomyces cerevisiae 3-phoshoglyceric acid kinase gene, yeast saccharomyces cerevisiae alpha factor gene and Ethanol in Saccharomyces cerevisiae desaturase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP) gene.

Control sequence can also be the polyadenylation sequence, namely effectively is connected in polynucleotide 3 ' terminal and sequence added the signal of polyadenylic acid residue to institute's transcript mRNA by host cell identification conduct when transcribing.Any polyadenylation sequence that has function in selected host cell can be used for the present invention.

The preferred polyadenylation sequence that is used for filamentous fungal host cell obtains from aspergillus oryzae TAKA amylase gene, aspergillus niger glucoamylase gene, Aspergillus nidulans anthranilic acid synthase gene, sharp sickle spore trypsin-like proteinase gene and aspergillus niger α Polyglucosidase gene.

Be used for the polyadenylation sequence description of yeast host cell in Guo and Sherman, 1995, Molecular Cellular Biology 15:5983-5990.

Control sequence can also be signal peptide coding region, and wherein said signal peptide coding region coding is connected in the aminoterminal aminoacid sequence of proteolytic enzyme and instructs coded proteolytic enzyme to enter the Secretory Pathway of cell.Encoding sequence 5 ' the end of polynucleotide can comprise coding region fragment with coding secreted protein enzyme inherently with the natural signal peptide coding region that is connected of translation reading frame.Alternatively, 5 ' of encoding sequence end can comprise encoding sequence is external signal peptide coding region.When encoding sequence does not comprise signal peptide coding region in natural situation, may need external signal peptide coding region.Alternatively, external signal peptide coding region can be replaced natural signal peptide coding region simply so that the secretion of Enhancin enzyme.Yet any signal peptide coding region that can instruct expressed proteolytic enzyme to enter the Secretory Pathway of selected host cell can be used for the present invention.

The effective signal peptide coding region that is used for filamentous fungal host cell obtains from aspergillus oryzae TAKA amylase gene, aspergillus niger neutral starch enzyme gene, aspergillus niger glucoamylase gene, rhizomucor miehei aspartic protease gene, lonely humicola lanuginosa (Humicola insolens) cellulose enzyme gene and pubescence humicola lanuginosa (Humicola lanuginosa) lipase gene.

Being used for the thin signal peptide coding region of yeast host obtains from yeast saccharomyces cerevisiae alpha factor gene and yeast saccharomyces cerevisiae invertase gene.Other useful signal peptide-coding region is described in Romanos etc., and 1992, the same.

The useful signal peptide-coding region that is used for bacterial host cell is from the signal coding sequence of genus bacillus NCIB 11837 maltose starch base because of, bacstearothermophilus α-amylase gene, Bacillus licheniformis subtilisin gene, Bacillus licheniformis beta lactamase gene, bacstearothermophilus neutral protease gene (nprT, nprS, nprM) and the acquisition of subtilis prsA gene.Other signal peptides are described in Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Control sequence can also be that coding is positioned at the N-terminal front peptide-coding region of proteolytic enzyme.The polypeptide that obtains is called proenzyme (proenzyme) or front polypeptide (or being called in some cases proenzyme (zymogen)).Front polypeptide is generally inactive and can be by catalytic cutting-out or autocatalysis cutting-out in propetide the past polypeptide is converted to ripe active polypeptide.Front peptide-coding region can obtain from bacillus subtilis alkali proteinase (aprE) gene, subtilis neutral protease (nprT) gene, yeast saccharomyces cerevisiae alpha factor gene, rhizomucor miehei aspartic protease gene and Myceliphthora thermophila laccase gene (WO 95/33836).

When signal peptide and propetide district all come across the N-terminal of subtilase, propetide district next-door neighbour's proteolytic enzyme aminoterminal and the signal peptide district is positioned at the aminoterminal in propetide district then.

It also is suitable adding the adjusting sequence that expression of polypeptides is regulated with respect to the host cell growth.The example of regulation system is those to chemical stimulation or physical stimulation (comprising the existence of modulating compound) thereby makes a response and cause the system that genetic expression is opened or closed.Regulation system in the prokaryotic system comprises lac, tac and trp operator gene system.In yeast, can use ADH2 system or GAL1 system.In filamentous fungus, TAKA α-amylase promotor, aspergillus niger glucoamylase promotor and aspergillus oryzae glucoamylase promotor can be used as the adjusting sequence.Other example of regulating sequence is those adjusting sequences that allow gene amplification.In eukaryotic system, these are regulated sequence and comprise the dihydrofolate reductase gene that can increase and the metallothionein gene that increases in the presence of methotrexate in the presence of heavy metals.In these cases, the polynucleotide of coded polypeptide effectively are connected with the adjusting sequence.

Expression vector

The invention still further relates to the recombinant expression vector that comprises nucleic acid construct of the present invention, promotor and transcription termination signal and translation termination signal.

Recombinant expressed year of nucleic acid construct that comprises code book invention enzyme can be any carrier that conveniently carries out the recombinant DNA operation.

The host cell that this carrier will import is usually depended in the selection of carrier.Therefore, carrier can be autonomously replicationg vector, namely copies the carrier that the outer entity of the karyomit(e) that does not rely on chromosome duplication exists, for example plasmid as carrier.Alternatively, carrier can be at a kind of carrier that imports the host cell rear section or all be integrated into the host cell gene group and together copy with karyomit(e) that carrier imports.

Carrier is preferably expression vector, and the dna sequence dna of code book invention enzyme is transcribed required extra fragments with DNA and effectively is connected in this carrier.Usually expression vector is derived from plasmid or viral DNA, perhaps comprises the composition of the two.Term " effectively connect " expression fragment is so arranged so that this their expectation function of class fragment Coordinated Play, for example transcribes initial from promotor and advances by the dna sequence dna of codase.

Promotor can be any dna sequence dna of performance transcriptional activity in selected host cell, and can be derived from the gene of the protein of coding and host cell homology or allos.

The suitable promotor example that is used for bacterial host cell comprises the promotor of bacstearothermophilus maltogenic amylase gene, bacillus licheniformis alpha amylase gene, bacillus amyloliquefaciens α-amylase gene, bacillus subtilis alkali proteinase gene or bacillus pumilus (Bacillus pumilus) xylosidase gene, perhaps phageλ PR or PL promotor or intestinal bacteria lac, trp or tac promotor.

If necessary, the dna sequence dna of code book invention enzyme effectively can also be connected with suitable terminator.

Recombinant vectors of the present invention also can further comprise the dna sequence dna that carrier is copied in the discussion host cell.

Carrier can also comprise selective marker, such as the gene of its product compensation host cell defective or coding to microbiotic such as kantlex, paraxin, erythromycin, tsiklomitsin, spectinomycin etc. or gene that heavy metal or weedicide are had resistance.

In order to instruct enzyme of the present invention to enter the Secretory Pathway of described host cell, can in recombinant vectors, provide secretory signal sequence (being also referred to as leader sequence, presequence former (prepro sequence) or presequence).Secretory signal sequence can be connected with the dna sequence dna of codase with correct reading frame.Secretory signal sequence is usually located at the 5' end of the dna sequence dna of codase.Secretory signal sequence can be the secretory signal sequence that normally is connected with enzyme or the gene that comes the another kind of secreted protein of own coding.

Be used for the dna sequence dna of code book invention enzyme respectively with promotor be connected the method that terminator and/or secretory signal sequence be connected wantonly or be used for by suitable pcr amplification scheme assemble the method for these sequences and be used for their insertions comprise copy or integrate the method in must the suitable carrier of information be those skilled in the art well-known (reference example such as Sambrook etc., the same).

Host cell

The invention still further relates to the recombinant host cell that comprises nucleic acid construct of the present invention.

Import host cell code book invention enzyme dna sequence dna for the discussion host for homology or allos.If described dna sequence dna and host cell homology, namely by the natural generation of this host cell, then described dna sequence dna is general effectively is connected with another promoter sequence or (if feasible) is connected with another kind of secretory signal sequence and/or terminator sequence in being different from this sequence natural surroundings.Term " homology " is intended to comprise that coding is for the dna sequence dna of discussion host living beings for natural enzyme.Term " allos " is intended to comprise the natural dna sequence dna of not expressing of host cell.Therefore, dna sequence dna can be from another kind of biological, and perhaps it is artificial synthesized sequence.

The host cell that DNA construct of the present invention or recombinant vectors import can be any cell that can produce enzyme of the present invention, the higher eucaryotic cells that it comprises bacterium, yeast, fungi and comprises plant.

The example that can produce the bacterial host cell of enzyme of the present invention when cultivating is gram positive bacterium, for example Bacillus strain is such as Bacillus subtillis, Bacillus licheniformis, bacillus lentus, bacillus brevis, bacstearothermophilus, Alkaliphilic bacillus, bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacillus lautus, bacillus megaterium or bacillus thuringiensis bacterial strain, especially bacillus lentus bacterial strain, perhaps streptomycete bacterial strain is such as muta lead mycillin or mouse ash streptomycete bacterial strain, perhaps Gram-negative bacteria such as intestinal bacteria.

The conversion of bacterium can realize (with reference to Sambrook etc., the same) in a manner known way by protoplast transformation, electroporation, combination or by competent cell.

When enzyme during at bacterium such as expression in escherichia coli, this enzyme generally can be used as insoluble particle (being called inclusion body) and stays in the tenuigenin or instructed by the bacterium secretion sequence and secrete to periplasmic space.In the previous case, with lysis and recovery particle and sex change, after this make the enzyme refolding by the dilution denaturing agent.Under latter event, can pass through smudge cells (as by supersound process or osmotic shock in order to discharge the periplasmic space content) obtain enzyme from periplasmic space, and reclaim enzyme.

When enzyme when gram positive bacterium is expressed in such as genus bacillus or streptomycete bacterial strain, enzyme can be trapped in the tenuigenin or by the bacterium secretion sequence and instruct secretion to the extracellular substratum.Under latter event, as described belowly can from substratum, obtain enzyme.

In another embodiment of the invention, fungal host cells is yeast cell." yeast " comprises ascosporogenous yeast (Endomycetale (Endomycetales)), produces sporidium order (basidiosporogenous) and belongs to the yeast (budding yeast genus (Blastomycetes)) of imperfect fungi as used herein.Because the classification of yeast may change in the future, for the purposes of the present invention, yeast should be such as Biology andActivities of Yeast (Skinner, F.A., Passmore, S.M. and Davenport, R.R., editor, Soc.App.Bacteriol.Symposium Series No.9,1980) describe and define.

In preferred embodiments, yeast host cell is mycocandida, Hansenula (Hansenula), genus kluyveromyces, Pichia, yeast belong, Schizosaccharomyces or inferior sieve yeast belong cell.

In more preferred, yeast host cell is saccharomyces carlsbergensis, yeast saccharomyces cerevisiae, saccharomyces diastaticus, Saccharomyces douglasii, Crewe not yeast, promise ground yeast or Saccharomyces oviformis cell.In another the most preferred embodiment, yeast host cell is Kluyveromyces lactis (Kluyveromyces lactis) cell.In another embodiment of selecting most, yeast host cell is Yarrowia lipolytica (Yarrowia lipolytica) cell.

In another preferred embodiment, fungal host cells is filamentous fungal cells." filamentous fungus " comprise Mycophyta (Eumycota) and oomycetes door (Oomycota) subphylum all fungies (such as Hawksworth etc., 1995, on seeing, define).Being characterized as of filamentous fungus has the mycelia wall that is made of chitin, Mierocrystalline cellulose, dextran, chitosan, mannosans and other complicated polysaccharide.Nourish and grow to prolong by mycelia and carry out and the carbon metabolism is that obligate is aerobic.Different therewith, yeast for example nourishing and growing of yeast saccharomyces cerevisiae is to be undertaken and the carbon metabolism can be fermentation by sprouting of unicellular thalline.

In addition the embodiment that is more preferably in, filamentous fungal host cell is such as Fusarium, the mould genus of top spore, Aspergillus, Humicola, Mucor, myceliophthora, Neurospora, Penicillium, Thielavia, Tolypocladium or the various cell of Trichoderma, but is not limited to this.

In most preferred embodiment, described filamentous fungal host cell is Aspergillus awamori, smelly aspergillus, aspergillus japonicus, Aspergillus nidulans, aspergillus niger, aspergillus oryzae cell.In another the most preferred embodiment, filamentous fungal host cell is bar spore shape sickle spore, Fusarium cerealis, Fusarium crookwellense, machete sickle spore, fusarium graminaria, the red sickle spore of standing grain, different spore sickle spore, albizzia sickle spore, sharp sickle spore, racemosus sickle spore, pink sickle spore, Williams Elder Twig sickle spore, colour of skin sickle spore, intends branch spore sickle spore, sulphur look Fusariumsp, Fusariumtorulosum, Fusarium trichothecioides, Fusarium venenatum cell.In addition the most preferred embodiment in, described filamentous fungal parent cell is Fusarium venenatum (Nirenberg sp.nov.) cell.In another the most preferred embodiment, described filamentous fungal host cell is that lonely humicola lanuginosa, pubescence humicola lanuginosa, rice black wool are mould, Myceliphthora thermophila, Neuraspora crassa, penicillium purpurogenum, Thielavia terrestris, trichoderma harziarum, mould, wooden mould, the wooden mould or viride cell of Li Shi of long handle of healthy and free from worry wood.

The fungal cell can transform in a manner known way by the method that comprises protoplastis formation, protoplast transformation and cell walls regeneration.The method that is applicable to transform the Aspergillus host cell is described in EP 238023 and Yelton etc., 1984, Proceedings of the National Academy of Sciences USA81:1470-1474.Be applicable to transform the various method of Fusarium and be described in Malardier etc., 1989, Gene 78:147-156 and WO 96/00787.Can use Becker and Guarente (at Abelson to the conversion of yeast, J.N. and Simon, M.I., editor, Guide to Yeast Genetics and MolecularBiology, Methods in Enzymology, the 194th volume, 182-187 page or leaf, Academic Press, Inc., among the New York); Ito etc., 1983, Journal of Bacteriology 153:163 and Hinnen etc., the method for describing among 1978, the Proceedings of the National Academy of Sciences USA 75:1920 is carried out.

Produce the method for proteolytic enzyme of the present invention

The invention still further relates to the method that produces proteolytic enzyme of the present invention, described method comprises:

A) inducing cultivation recombinant host cell of the present invention under the condition that produces proteolytic enzyme, and

B) reclaim proteolytic enzyme.

When the expression vector conversion of the dna sequence dna that will comprise codase enters in the heterologous host cell, might realize the heterologous recombination production of enzyme of the present invention.

Therefore can produce highly purified protease composition, it is characterized in that not containing homology impurity.

In the context of the invention, homology impurity means any impurity (for example other polypeptide except enzyme of the present invention) from homologous cell, and wherein said homologous cell is the cell that begins to obtain enzyme of the present invention most.

Substratum that be used for to cultivate institute's transformed host cell can be to be suitable for any conventional medium that institute's host cell of discussing is grown.Expressed proteolytic enzyme can be secreted into easily in the substratum and can be by well-known method from wherein reclaiming, wherein said method comprise through centrifugal or filter from substratum isolated cell, by salt for example the ammonium sulfate precipitation mode precipitate protein component in the substratum, be chromatography method for example ion exchange chromatography, affinity chromatography etc. subsequently.

The purposes of proteolytic enzyme of the present invention

Proteolytic enzyme of the present invention can be used for being particularly useful for detergent industry in numerous industrial application.Therefore, the invention still further relates to the cleaning compositions or the detergent composition that comprise proteolytic enzyme of the present invention, preferred laundry composition or wash the dish composition.

The detergent composition that comprises proteolytic enzyme of the present invention

Usually, cleaning compositions and detergent composition are described through abundant in this area, and can be with reference to WO 96/34946 to suitable cleaning compositions and further describing of detergent composition; WO97/07202; WO 95/30011.

And embodiment herein proves that the stability of proteolytic enzyme of the present invention in washing composition improves.

Detergent composition

Enzyme of the present invention can be added into a kind of composition that in cleaning compositions or the detergent composition and therefore becomes them.

Detergent composition of the present invention for example can be mixed with hand washing or machine washing of clothes detergent composition (it comprises the fabric softener composition that the laundry additive composition that is applicable to the dirty fabric of pre-treatment and rinsing add), perhaps be mixed with the detergent composition for general household hard surface cleaning operation, perhaps be mixed with for manual and use or the machine detergent composition of washing the dish operation.

A particular aspects, the invention provides the detergent additives that comprises proteolytic enzyme of the present invention.Detergent additives and detergent composition can comprise one or more other enzymes, such as another kind of proteolytic enzyme, lipase, at, amylase, carbohydrase, cellulase, polygalacturonase, mannonase arabinase, galactanase, zytase, oxydase such as laccase and/or peroxidase.

Usually, the feature of selected then enzyme should be compatible with selected washing composition (for example best pH, with consistency of other enzyme or non-enzyme composition etc.), and enzyme should exist with significant quantity.

Proteolytic enzyme: the adequate proteins enzyme comprises those animal-origins, plant origin or microbe-derived proteolytic enzyme.The proteolytic enzyme in preferred microorganism source.Mutant chemically modified or the protein engineering transformation is included.Proteolytic enzyme can be serine protease or metalloprotease, preferred alkaline microbial protease or trypsin-like proteolytic enzyme.The example of Sumizyme MP is subtilisin, especially those are derived from the subtilisin of bacillus, such as subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168 (describing in WO 89/06279).The example of trypsin-like proteolytic enzyme is trypsin such as pig or Niu Laiyuan) and the Fusarium proteolytic enzyme in WO 89/06270 and WO94/25583, described.

The example of useful proteins enzyme is the variant of describing in WO 92/19729, WO 98/20115, WO 98/20116 and WO 98/34946, especially has one or more alternative variants such as upper/lower positions: 27th, 36,56,76,87,95,96,97,98,99,100,101,102,103,104,120,123,159,167,170,206,218,222,224,232,235,236,245,248,252 and 274 (BPN ' numbering).

Preferably can comprise Alcalase by the commercial proteolytic enzyme that obtains ^TM, Savinase ^TM, Primase ^TM, Duralase ^TM, Esperase ^TM, Kannase ^TM(Novozymes A/S), Maxatase ^TM, Maxacal ^TM, Maxapem ^TM, Properase ^TM, Purafect ^TM, Purafect OxP ^TM, FN2 ^TMAnd FN3 ^TM(Genencor International Inc.).

Lipase: suitable lipase comprises the lipase of those bacterial origins or originated from fungus.Mutant chemically modified or the protein engineering transformation is included.The example of useful lipase comprises from the lipase of Humicola (synonym thermophilic fungus genus) such as the pubescence humicola lanuginosa of describing in EP 258 068 and EP 305 216 or the lonely humicola lanuginosa of describing in WO 96/13580; Pseudomonas Lipases Tathagata is from Pseudomonas alcaligenes (P.alcaligenes) or pseudomonas pseudoalcaligenes (P.pseudoalcaligenes) (EP 218272), pseudomonas cepacia (P.cepacia) (EP 331 376), Pseudomonas stutzeri (P.stutzeri) (GB1,372,034), Pseudomonas fluorescens (P.fluorescens), pseudomonas kind SD 705 bacterial strains (Pseudomonas sp.strain SD 705) (WO 95/06720 and WO 96/27002), the lipase of P.wisconsinensis (WO 96/12012); Genus bacillus lipase is as from Bacillus subtillis (Dartois etc., (1993), Biochemica et BiophysicaActa, 1131,253-360), the lipase of bacstearothermophilus (JP 64/744992) or bacillus pumilus (WO 91/16422).

Other examples are those lipase Variants of describing as in WO 92/05249, WO 94/01541, EP 407 225, EP 260 105, WO 95/35381, WO 96/00292, WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079 and WO 97/07202.

Preferably can comprise Lipolase by the commercial lipase that obtains ^TMAnd LipolaseUltra ^TM(Novozymes A/S).

Amylase: suitable (α and/or β) amylase comprises the amylase of those bacterial origins or originated from fungus.Mutant chemically modified or the protein engineering transformation is included.Amylase for example comprises from genus bacillus as more being described in detail in GB 1,296, the α-amylase that the Bacillus licheniformis particular strain in 839 obtains.

Useful diastatic example is the variant of describing in WO 94/02597, WO 94/18314, WO 96/23873 and WO 97/43424, especially has one or more alternative variants such as upper/lower positions: 15th, 23,105,106,124,128,133,154,156,181,188,190,197,202,208,209,243,264,304,305,391,408 and 444.

Can the commercial amylase that obtains be Duramyl ^TM, termamyl ^TM, Fungamyl ^TMAnd BAN ^TM(Novozymes A/S), Rapidase ^TMAnd Purastar ^TM(from Genencor International Inc.).

Cellulase: suitable cellulase comprises the cellulase of those bacterial origins or originated from fungus.Mutant chemically modified or the protein engineering transformation is included.Suitable cellulase comprises the cellulase from bacillus, Rhodopseudomonas, Humicola, Fusarium, Thielavia, the mould genus of top spore, for example be disclosed in US 4,435,307, US 5,648, and 263, US 5,691,178, US 5,776,757 and WO 89/09259 in the fungal cellulase that is produced by lonely humicola lanuginosa, Myceliphthora thermophila and sharp sickle spore.

Especially the cellulase that is fit to is alkalescence or the neutral cellulase that helps protect look.The example of this type of cellulase is the cellulase of describing in EP 0495257, EP 0531372, WO 96/11262, WO 96/29397, WO98/08940.Other examples are those cellulase variants of describing in WO 94/07998, EP 0 531 315, US 5,457,046, US 5,686,593, US 5,763,254, WO 95/24471, WO 98/12307 and PCT/DK98/00299.

Can comprise Celluzyme by the commercial cellulase that obtains ^TMAnd Carezyme ^TM(NovozymesA/S), Clazinase ^TM, and Puradax HA ^TM(Genencor International Inc.) and KAC-500 (B) ^TM(Kao Corporation).

Peroxidase/oxydase: suitable peroxidase/oxydase comprises the peroxidase/oxydase of those plants, bacterium or originated from fungus.Mutant chemically modified or the protein engineering transformation is included.The example of useful peroxidase comprises their variant of describing such as the peroxidase of Coprinus cinereus (C.cinereus) and in WO 93/24618, WO 95/10602 and WO 98/15257 from Coprinus (Coprinus).

Comprise the independent additive of one or more enzymes or comprise the combined additive of all these enzymes by interpolation by interpolation, make to comprise detergent enzyme in the detergent composition.Detergent additives of the present invention (being independent additive or combined additive) can be mixed with particle, liquid, slurry night etc.Preferred detergent additives preparation is particle (especially being non-dirt shape particle), liquid (especially stabilising liq) or slurries.

The particle of non-dirt shape can be for example according to US 4,106, disclosed such produce and optionally by the methods known in the art dressing in 991 and 4,661,452.The example of wax coating material be molecular-weight average be 1000-20000 poly-(oxyethane) product (polyoxyethylene glycol, PEG); Ethoxylized nonylphenol with 16-50 ethylene oxide unit; In alcohol, comprise 12-20 carbon atom and have the ethoxylized fatty alcohol of 15-18 ethylene oxide unit; Fatty Alcohol(C12-C14 and C12-C18); The direactive glyceride of lipid acid and lipid acid, two glyceryl ester and Witepsol W-S 55.The example that is applicable to form by fluidization the coating material of film provides in GB143591.Liquid enzyme formulation can carry out stabilizing treatment by adding polyvalent alcohol such as propylene glycol, sugar or sugar alcohol, lactic acid or boric acid according to establishment method.Enzyme through protection can be according to disclosed method preparation in EP 238,216.

Detergent composition of the present invention can be any form that makes things convenient for, such as bar-shaped, sheet, powder, particulate state, pasty state or liquid.Liquid washing agent can be liquid, aqueous, generally contains up to 70% water and the organic solvent of 0-30%, or on-aqueous liquid.

Detergent composition generally comprises one or more tensio-active agents, and described tensio-active agent can be nonionogenic tenside and/or anion surfactant and/or cats product and/or the zwitterionics that comprises semi-polarity.The tensio-active agent level is generally the 0.1%-60% of weight.

When in cleaning composition, comprising anion surfactant, described washing composition generally comprises about 1% to about 4% anion surfactant, for example linear alkylbenzene sulphonic acid, alhpa olefin sulphonate, alkyl-sulphate (aliphatic alcohol sulfate), alcohol ethoxy vitriol, secondary alkyl sulfonate, α thia fatty acid methyl esters, alkyl or alkenyl succsinic acid or soap.

When in cleaning composition, comprising nonionogenic tenside; described washing composition generally comprises about 0.2% to about 40% nonionogenic tenside, for example the N-acyl group N-alkyl derivative of alcohol ethoxylate, nonyl phenol ethoxylate, alkyl polyglucoside, alkyl dimethyl amine oxide, ethoxylated fatty acid single ethanol amide, fatty monoethanol amide, polyhydroxy alkyl fatty amide or glycosamine (" glucamide (glucamides) ").

Washing composition can comprise washing assistant or the complexing agent of 0-65%, such as zeolite, diphosphate, triphosphate, phosphonate, carbonate, Citrate trianion, nitrilotriacetic acid(NTA), ethylenediamine tetraacetic acid (EDTA), diethylene triaminepentaacetic acid(DTPA), alkyl or alkenyl succinic, soluble silicate or layered silicate (for example from Hoechst SKS-6).

Washing composition can comprise one or more polymkeric substance.The example of polymkeric substance is carboxymethyl cellulose, polyvinylpyrrolidone, polyoxyethylene glycol, polyvinyl alcohol, polyvinyl pyridine N-oxide, polyvinyl imidazole, polycarboxylate such as polyacrylic ester, toxilic acid/acrylic copolymer and lauryl methacrylate(LMA)/acrylic copolymer.

Washing composition can comprise the bleaching system that contains hydrogen peroxide cource such as perborate or percarbonate, and wherein said hydrogen peroxide cource can mix with the bleaching activator that forms peracid such as tetra acetyl ethylene diamine or nonanoyl hydroxy benzene sulfonate.Alternatively, bleaching system can comprise for example peroxy acid of acid amide type, imide type or sulfone type.

Enzyme in the detergent composition of the present invention (class) can use conventional stablizer to stablize, stablizer such as polyvalent alcohol be propylene glycol or glycerine, sugar or sugar alcohol, lactic acid, boric acid or boric acid derivatives such as fragrant boric acid ester or phenyl-boron dihydroxide derivative such as 4-formylphenyl boric acid for example, and composition can be according to preparation described in WO92/19709 and WO 92/19708.

Washing composition can also comprise for example fabric regulator of other conventional washing composition composition, comprises that clay, profoamer, suds suppressor, sanitas, soil-suspending agent, dirt-proof be stained with agent, dyestuff, sterilant, white dyes, hydrotropic agent, tarnish inhibitor or spices again.

Consider at present according to be equivalent to every liter of washing liq 0.01-100 milligram zymoprotein, preferred every liter of washing liq 0.05-5 milligram zymoprotein, especially the amount of every liter of washing liq 0.1-1 milligram zymoprotein adds any enzyme, enzyme especially of the present invention in detergent composition.

Enzyme of the present invention can additionally join disclosed detergent formulation among the WO 97/07202.

The present invention further describes in detail in following embodiment, and described embodiment in no case limits scope of the presently claimed invention.

In detergent composition, the component of abbreviation sign has following implication:

LAS: straight chain C ₁₂Sodium alkyl benzene sulfonate

TAS: Tallow, beef sodium alkyl sulfate

XYAS:C _1X-C _1YSodium alkyl sulfate

SS: molecular formula is the sad secondary soap surfactant of 2-butyl

25EY: with the C that is mainly of the ethylene oxide condensation of average Y mole ₁₂-C ₁₅Straight chain primary alcohol

45EY: with the C that is mainly of the ethylene oxide condensation of average Y mole ₁₄-C ₁₅Straight chain primary alcohol

XYEZS: every mole of C with the ethylene oxide condensation of average Z mole _1X-C _1YSodium alkyl sulfate

Nonionogenic tenside: by the C of BASF GmbH with trade(brand)name Plurafax LF404 sale ₁₃-C ₁₅Mixed ethoxylated/propoxylated fatty alcohol, it has average degree is that 3.8 oxyethylation and average degree are 4.5 oxypropylation

CFAA:C ₁₂-C ₁₄Alkyl N-methyl glucose amide

TFAA:C ₁₆-C ₁₈Alkyl N-methyl glucose amide

Silicate: amorphous sodium silicate (SiO ₂: Na ₂O ratio=2.0)

NaSKS-6: molecular formula is δ-Na ₂Si ₂O ₅The crystalline state layered silicate

Carbonate: anhydrous sodium carbonate

Phosphoric acid salt: tripoly phosphate sodium STPP

MA/AA: toxilic acid/vinylformic acid is the multipolymer of 1:4, and its molecular-weight average is approximately 80,000

Polyacrylate: by the polyacrylate homopolymer that BASF GmbH sells with trade(brand)name PA30, its molecular-weight average is 8,000

Wessalith CS: the molecular formula with original particle size size of 1-10 micron is Na ₁₂(AlO ₂SiO ₂) ₁₂.27H ₂The hydrated aluminium silicate sodium of O

Citrate trianion: citrate trisodium dihydrate

Citric acid: citric acid

Perborate: anhydrous sodium perborate monohydrate SYNTHETIC OPTICAL WHITNER, empirical formula are NaBO ₂.H ₂O ₂

PB4: anhydrous sodium perborate tetrahydrate

Percarbonate: empirical formula is 2Na ₂CO ₃.3H ₂O ₂Anhydrous SPC-D SYNTHETIC OPTICAL WHITNER

TAED: tetra acetyl ethylene diamine

CMC: Xylo-Mucine

DETPMP: diethylene triamine penta(methylene phosphonic acid), sold with trade(brand)name Dequest2060 by Monsanto

PVP: polyvinyl pyrrolidone polymers

EDDS: the EDDS of sodium-salt form, [S, S] isomer

Suds suppressor: 25% paraffin (fusing point is 50 ℃), 17% water drain silica, 58% paraffin oil

Granular suds suppressing agent: 12% siloxanes/silicon-dioxide, 18% Stearyl alcohol, 70% particle form starch

Vitriol: anhydrous sodium sulphate

HMWPEO: high molecular weight polyethylene oxide

TAE25: Tallow, beef alcohol ethoxylate (25)

Washing composition example I

Particulate state clean fabric composition of the present invention can be prepared as follows:

Component	%
		Straight chain C ₁₂Sodium alkyl benzene sulfonate	6.5
Sodium sulfate	15.0
		Wessalith CS	26.0
Sodium nitrilo triacetate	5.0
		Enzyme	0.1
PVP	0.5
		TAED	3.0
Boric acid	4.0
		Perborate	18.0
Phenolsulfonate	0.1
		Minor materials (Minors)	To 100

Washing composition example II

Tight particulate state clean fabric composition of the present invention (density 800g/l) can be prepared as follows:

Component	%
		45AS	8.0
25E3S	2.0
		25E5	3.0
25E3	3.0
		TFAA	2.5
Wessalith CS	17.0
		NaSKS-6	12.0
Citric acid	3.0

Carbonate	7.0
		MA/AA	5.0
CMC	0.4
		Enzyme	0.1
TAED	6.0
		Percarbonate	22.0
EDDS	0.3
		The particulate state suds suppressor	3.5
Water/minor materials	To 100

Washing composition example III

The particulate state clean fabric composition that is particularly useful for washing DYED FABRICS of the present invention can be prepared as follows:

Component	%	%
			LAS	10.7	-
TAS	2.4	-
			TFAA	-	4.0
45AS	3.1	10.0
			45E7	4.0	-
25E3S	-	3.0
			68E11	1.8	-
25E5	-	8.0
			Citrate trianion	15.0	7.0
Carbonate	-	10.0
			Citric acid	2.5	3.0
Wessalith CS	32.1	25.0
			Na-SKS-6	-	9.0
MA/AA	5.0	5.0
			DETPMP	0.2	0.8
Enzyme	0.10	0.05
			Silicate	2.5	-
Vitriol	5.2	3.0
			PVP	0.5	-
Poly-(4-vinylpyridine)-N-oxide compound/ethene imidazoles

Multipolymer with V-Pyrol RC	-	0.2
			Perborate	1.0	-
Phenolsulfonate	0.2	-
			Water/minor materials	To 100	To 100

Washing composition example IV

The particulate state clean fabric composition of of the present invention can providing " softening in the washing process " ability can be prepared as follows:

Component	%	%
			45AS	-	10.0
LAS	7.6	-
			68AS	1.3	-
45E7	4.0	-
			25E3	-	5.0
Cocounut oil alkyl-dimethyl-hydroxyl-ethyl ammonium chloride	1.4	1.0
			Citrate trianion	5.0	3.0
Na-SKS-6	-	11.0
			Wessalith CS	15.0	15.0
MA/AA	4.0	4.0
			DETPMP	0.4	0.4
Perborate	15.0	-
			Percarbonate	-	15.0
TAED	5.0	5.0
			Terre verte	10.0	10.0
HMWPEO	-	0.1
			Enzyme	0.10	0.05
Silicate	3.0	5.0
			Carbonate	10.0	10.0
The particulate state suds suppressor	1.0	4.0
			CMC	0.2	0.1
Water/minor materials	To 100%	To 100%

Washing composition example V

The present invention crosses dirty liquid body clean fabric composition and can be prepared as follows:

Component	%	%
			LAS acid form	-	25.0
Citric acid	5.0	2.0
			25AS acid form	8.0	-
25AE2S acid form	3.0	-
			25AE7	8.0	-
CFAA	5.0	-
			DETPMP	1.0	1.0
Lipid acid	8	-
			Oleic acid	-	1.0
Ethanol	4.0	6.0
			Propylene glycol	2.0	6.0
Enzyme	0.10	0.05
			Cocounut oil alkyl dimethyl hydroxyethyl ammonium chloride	-	3.0
Terre verte	-	5.0
			PVP	2.0	-
Water/minor materials	To 100	To 100

Powdered automatic washing dish composition I

Nonionogenic tenside	0.4-2.5%
		Starso	0-20%
Sodium disilicate	3-20%
		Tri sodium Phosphate	20-40%
Yellow soda ash	0-20%
		Sodium peroxoborate	2-9%
Tetra acetyl ethylene diamine (TAED)	1-4%
		Sodium sulfate	5-33%
Enzyme	0.0001-0.1%

Powdered automatic washing dish compositions II

Powdered automatic washing dish composition III

Nonionogenic tenside	0.5-2.0%
		Sodium disilicate	25-40%
Trisodium Citrate	30-55%
		Yellow soda ash	0-29%
Sodium bicarbonate	0-20%
		The Sodium peroxoborate monohydrate	0-15%
Tetra acetyl ethylene diamine (TAED)	0-6%
		Toxilic acid/acrylic copolymer	0-5%
Clay	1-3%
		Polyamino acid	0-20%
Sodium polyacrylate	0-8%
		Enzyme	0.0001-0.1%

Powdered automatic washing dish composition IV

Powdered automatic washing dish composition V

Nonionogenic tenside	1-7%
		Sodium disilicate	18-30%
Trisodium citrate	10-24%
		Yellow soda ash	12-20%
Single persulphate (2 KHSO ₅.KHSO ₄.K ₂SO ₄)	15-21%
		Bleaching stibilizer	0.1-2%
Toxilic acid/acrylic copolymer	0-6%
		Diethylenetriamine pentaacetic acid, five sodium-salt	0-2.5%
Enzyme	0.0001-0.1%
		Sodium sulfate, water	Balance

Powdered and liquid with cleansing surfactants system is washed dish composition VI

On-aqueous liquid automatic washing dish composition VII

The nonionogenic tenside of liquid (for example alcohol ethoxylate)	2.0-10.0%
		Alkalimetal silicate	3.0-15.0%
Alkali metal phosphate	20.0-40.0%
		Be selected from the liquid vehicle of senior dibasic alcohol, polyoxyethylene glycol, polyoxide, glycol ether	25.0-45.0%
Stablizer (for example phosphoric acid and C ₁₆-C ₁₈The partial ester of alkanol)	0.5-7.0%
		Suds suppressor (for example silicone)	0-1.5%
Enzyme	0.0001-0.1%

On-aqueous liquid wash dish composition VIII

The liquid automatic washing dish composition IX of thixotropy

Liquid automatic washing dish composition X

Alcohol ethoxylate	0-20%
		Fatty sulfonate	0-30%
Sodium lauryl sulphate	0-20%
		APG	0-21%
Oleic acid	0-10%
		One hydration sodium disilicate	18-33%
Sodium citrate dehydrate	18-33%
		Sodium stearate	0-2.5%
Sodium perborate monohydrate	0-13%
		Tetra acetyl ethylene diamine (TAED)	0-8%
Toxilic acid/acrylic copolymer	4-8%
		Enzyme	0.0001-0.1%

The liquid automatic washing dish composition XI that contains shielded bleaching particle

XII: such as the automatic washing dish composition described in I, II, III, IV, VI and the X, wherein replace perborate with percarbonate.

The XII I: such as the automatic washing dish composition described in I-VI, it also contains Mn catalyst.Mn catalyst can be for example " Efficient manganese catalysts for low-temperature bleaching ", Nature, (1994), one of compound described in 369, the 637-639.

Materials and methods

Fungal bacterial strain fusarium solanae (Fusarium solani)

Aspergillus oryzae strain BECh2 (WO 00/39322)

Expression vector pCaHj483 (WO 98/00529)

Aspergillus expression construct pMStr59 (example VI)

The protein-active enzyme assay

The AZCL-method for casein:

Substrate: AZCL-casein (from the casein of the crosslinked of Megazyme and dyeing, article No. I-AZCAS)

Temperature: controlled

Measure damping fluid:

● for the pH spectrogram:

О 100mM succsinic acid,

О 100mM HEPES，

О 100mM CHES，

О 100mM CABS，1mM CaCl ₂，

О 150mM KCl，

О 0.01%Triton X-100

Be adjusted to pH value 3.0 with HCl or NaOH; 4.0; 5.0; 6.0; 7.0; 8.0; 9.0; 10.0 and 11.0.

О for other: the 0.1M borax, pH 9.0.

The step that is used for the AZCL-method for casein:

1) stirs gently and 0.02g AZCL-casein is suspended in 10.0ml measures damping fluid.

2) be transferred to this suspension of 200 microlitres in the pipe and place ice bath, then 40 microlitre proteolytic enzyme samples (diluting in assay buffer) are added in the substrate pipe.

3) be transferred to by the pipe that will contain substrate samples and enzyme sample and begin test in the temperature mixing tank, wherein the temperature mixing tank was being measured under the temperature preheating at least 5 minutes.

4) pipe was hatched 20 minutes with 1200 rev/mins on the temperature mixing tank.

5) pipe is transferred in the ice bath and stops hatching.

6) then with pipe in refrigerated centrifuge centrifugal several minutes.

7) measure OD ₅₉₅, as the standard of measurement of relative protease activity.

The step that is used for the DMC/TNBS assay method:

The preparation of substrate and reagent:

О DMC substrate solution:

With 0.075g DMC (dimethyl casein, obtain from Novo Nordisk A/S) be dissolved in 10ml and measure damping fluid (with identical in the AZCL-method for casein), regulate pH with HCl or NaOH, be used as the DMC substrate solution with 0.45 micron membrane filtration and this filtrate.

О TNBS solution:

200mM CHES (obtaining from Sigma) damping fluid with 5ml pH 9.0 dilutes 17 microlitre 1MTNBS (obtaining from Fluka) solution, keeps in Dark Place on ice.

Step:

1) 40 microlitre DMC substrate solutions is transferred to 96 hole microtiter plates, adds subsequently 20 microlitre proteolytic enzyme samples, then fully mix and keep ice bath.

2) begin in the temperature mixing tank to measure by microtiter plate is transferred to, wherein the temperature mixing tank was being measured under the temperature preheating at least 5 minutes.

3) pipe was hatched 20 minutes with 600 rev/mins on the temperature mixing tank.

4) microtiter plate is moved to ice bath and stop hatching, add subsequently 60 microlitre TNBS solution, in the dark in keeping 15 minutes on ice.

5) measure OD ₄₀₅Standard of measurement as relative protease activity.

Inhibition

Streptomyces subtilisin inhibition (SSI) by Novozymes A/S preparation

Chymotrypsin protein enzyme inhibitor-2 (CI-2) by Novozymes A/S preparation

Disodium EDTA (EDTA) from USB life science

The p-Nitroaniline chromogenic substrate

Suc-AAPF-pNA(Sigma S-7388)

Suc-AAPE-pNA(BACHEM L-1710)

Suc-GGF-pNA(Sigma S-1899)

BA-pNA (benzoyl-DL-arginine, Sigma B-4875)

Suc-FGL-pNA(Sigma S-6768)

Example I: be used for the cultivation of the fungal bacterial strain of enzyme purification and gene clone

The fusarium solanae fungal bacterial strain that comprises code book invention proteinase gene fragment is cultivated concentrated growth at WB.

Each 500ml jolting culturing bottle:

● 30g wheat bran, and

● the following solution of 45ml:

О 0.18g yeast extract,

О 0.045g KH2PO4，

О 0.0225g MgSO4.7H2O，

О 0.675g glucose, the 45ml tap water,

121 ℃ of autoclavings 30 minutes kept 7 days at 25 ℃.

By adding about 150ml aqua sterilisa and using the glass stick of sterilization to stir, then 4 ℃ of placements are spent the night and are carried out the extraction of enzyme.Collect to filter and centrifugal after final supernatant liquor and be used as the crude protein enzyme sample that is further purified.

Then the WB substratum that directly 10g fermented goes in the cleaning plastics bag, and is immediately freezing with the results mycelium in liquid nitrogen.Freezing mycelium is stored in-80 ℃ of refrigerators until be used for the RNA extraction.

Example II: purifying protein enzyme from nutrient solution

Bacterial strain supernatant liquor described in the example I of 4000ml is used for protease purification.Total protein ammonium sulfate precipitation (80% saturation ratio).

After centrifugal, will precipitate and heavily be dissolved in the 100ml 25mM phosphate buffered saline buffer (pH6.0), then dialyse with same buffer.

The sample of will dialysing filters with 0.45 micron filter, then is added on the purification column.The sample final volume is 140ml.

The sample that filters is loaded on the 38ml SP agarose FF post (from Phamacia) of the phosphate buffered saline buffer balance of 25mM pH6.0, and with LINEAR N aCl gradient (0-0.3M) elute protein.At pH 9.0, in the situation that suppresses or do not suppress with SSI in advance, the analytical column fraction is to the caseic protease activity of AZCL-.

To have the protease activity fraction that is not suppressed by SSI mixes.Then with mixing solutions 3k film (from Amicon) ultrafiltration, concentrated solution is added to the Tris-HCl through 25mM, on 180mlSephacryl 100 posts of pH7.4 balance (from Phamacia).Use the same buffer elute protein.

At protease activity property testing (suppress in advance or do not suppress in advance with SSI with SSI) afterwards, have the protease activity fraction by the SDS-PAGE analysis, and will contain the fraction mixing of purifying protein enzyme and be used as sample characteristic description (seeing EXAMPLE IV).

EXAMPLE III: the gene clone of proteolytic enzyme

Use the gene fragment of RT-PCR technology clones coding proteolytic enzyme of the present invention.

The extraction of total RNA:

Use RNeasy Mini test kit (QIAGEN, catalog number (Cat.No.) 74904) from freezing mycelium, to extract total RNA.Total RNA extracts from the 100mg mycelium of the described bacterial strain of example I.

Be used for the design of clone's Auele Specific Primer:

Conservative region based on fungi known trypsinase dna sequence dna is designed for from Fusarium and Relative Fungi by the tryptic Auele Specific Primer of pcr amplification.Primer sequence (CDS primer) is shown in SEQ ID NO:5.

By using 3 ' RACE test kit clone proteolytic enzyme total length of the present invention:

3 ' RACE system (GIBCO, catalog number (Cat.No.) 18373-019) is for the synthesis of the cDNA of proteolytic enzyme of the present invention.With about 5 milligrams of total RNA as template and with first chain of Adapter Primer (being provided by 3 ' RACE system) for the synthesis of cDNA.Then use the cDNA of specific C DS-primer amplification proteolytic enzyme of the present invention.

The PCR product that uses the CDS primer amplification obtains approximately ~ specific band of 1kb fragment through gel analysis, reclaim this product from the 1%LMP sepharose, then by hatching and use subsequently PCR Preps dna purification system (Promega, catalog number (Cat.No.) A7170) purifying at 70 ℃.Purifying fragment and pGEM-T carrier (Promega, catalog number (Cat.No.) A3600) connect, and then the 2-4 microlitre are connected product and transform the efficient competent cell of 50 microlitre JM109 by " heat-shocked " method.

To transform nutrient solution and coat on the LB flat board that contains penbritin/IPTG/X-Gal, and with these flat boards in 37 ℃ of overnight incubation.

By indicating dithering and the bacterium colony PCR Screening and Identification recombinant clone on the flat board.Positive colony is inoculated in 3ml LB liquid nutrient medium and in 37 ℃ of vibrations (about 250 rev/mins) overnight incubation.Behind centrifugal 5 minutes sedimentation cells of 10,000g, use that preparing dna purification system (Promega, catalog number (Cat.No.) A7100) prepares the plasmid sample from cell precipitation in a small amount.At last, use BigDye Terminator CycleSequencing Ready Reaction test kit (PE) by the ABI377 sequenator plasmid to be checked order, and successfully obtain the full length sequence of this gene.

EXAMPLE IV: the feature description of proteolytic enzyme of the present invention

Suppress test:

Comprise that by use the different inhibitions of SSI, CI2 and EDTA test the proteolytic enzyme of the present invention (seeing example II) of purifying in the AZCL-method for casein, with disclosed trypsin-like proteolytic enzyme in WO 94/25583 and

In contrast.The results are shown in Fig. 1.

The activity of proteolytic enzyme of the present invention is not suppressed by SSI, CI-2 and EDTA, this means that this proteolytic enzyme may be the non-subtilisin in the serine stretch protein enzyme family.

Substrate specificity:

The substrate specificity of proteolytic enzyme of the present invention comprises the different pNA substrates test of AAPF, AAPE, GGF, BA and FGL by use, with

With disclosed trypsin-like proteolytic enzyme in WO 94/25583 as reference.The results are shown in Fig. 2.

Described proteolytic enzyme has identical substrate specificity with disclosed trypsin-like proteolytic enzyme in WO 94/25583 and it is different from subtilisin

The temperature spectrogram:

The temperature spectrogram of proteolytic enzyme of the present invention uses aforesaid AZCL-method for casein to obtain.The results are shown in Fig. 3, can find out described enzyme 15 ℃ to 70 ℃ have activity and optimum temps at 40 ℃ near 50 ℃.

The pH spectrogram:

The pH spectrogram of proteolytic enzyme of the present invention uses at the described AZCL-method for casein of " protease activity determination method " part and DMC assay method and obtains.

The results are shown in Fig. 5 (AZCL method for casein) and Fig. 4 (DMC assay method).

Described proteolytic enzyme has optimum activity at pH3 to pH11 show activity and near pH 9.In two kinds of assay methods, until pH 11 proteolytic enzyme all show high reactivity.

PH-stability:

For the pH stability test, 15 microlitre enzyme samples and 200

microlitre pH

3,4,5,6,7,8,9,10,11 damping fluid are mixed and hatched 2 hours at 37 ℃.Subsequently, use the as above described AZCL-method for casein mensuration of " protease activity determination method " part enzymic activity.The results are shown in Fig. 6.Can find out that proteolytic enzyme of the present invention is stable in the wide pH scope of pH 4 to pH 10.

EXAMPLE V: the stability in washing composition

Proteolytic enzyme (is dissolved in the 1.4g/l in the deionized water with deionized water dilution and with Attack detergent solution from KAO, 6 ° of dH (CaCl2:MgCl22:1), not heat inactivation) mix to produce the washing composition final concentration and relevant protease content of 0.7g/l (3 ° of dH).Be that the proteolytic enzyme dosage of 25-50mA/ml is for normal corresponding to OD280 in the mixture.Repeat three times.

Mixture hatched in 20 ℃, 25 ℃, 30 ℃ and 35 ℃ then in the DMC/TNBS assay method, measured actively in 30 minutes, perhaps mixture is measured as a reference immediately.

Residual activity shown in the following table is calculated as the activity of comparing the sample of storing with the reference sample activity.

Can find out that such as above data proteolytic enzyme of the present invention is compared with disclosed proteolytic enzyme in WO 9425583 aspect stable and demonstrated significantly improvement.

Example VI is expressed the trypsinase from fusarium solanae in aspergillus oryzae

The coding tryptic nucleotide sequence of fusarium solanae (SEQ ID NO:1) is used for the primer of the described gene of design pcr amplification, has added at the end of described primer to be beneficial to the suitable restriction site that the clone enters expression vector and (to see SEQ ID NO:3 and SEQ ID NO:4; Primer sequence).

Use Pfu Turbo archaeal dna polymerase (Stratagene, La Jolla, California, the U.S.) and according to manufacturer specification and use that cDNA carries out pcr amplification as template described in the EXAMPLE III, annealing temperature is 58 ℃ and 1 minute extension time, 30 circulations.Obtain single PCR product, with product with the restricted cutting of XhoI and be cloned among the aspergillus expression vector pCaHj483 (WO 98/00529), this carrier the Application standard technology with NruI and the restricted cutting of XhoI.Expression vector pCaHj483 contains the sequence of selecting and breeding and the sequence of selecting and expressing in aspergillus in intestinal bacteria.

Particularly, the selection in aspergillus is by the help of the amdS gene of Aspergillus nidulans, and this amdS gene allows to use ethanamide as only nitrogen source.Expression in aspergillus is by from the mediation of neutral starch enzyme II (NA2) promotor of aspergillus niger, this promotor with merge from 5 ' leader sequence of triose-phosphate isomerase (tpi) encoding gene of Aspergillus nidulans with from the encoding gene terminator of the amyloglucosidase of aspergillus niger.Measure the sequence comparison to confirm not introduce mistake during the pcr amplification with the trypsinase encoding gene order-checking of resulting aspergillus expression construct pMStr59 and with sequence with previous.

The Application standard technology (Christensen, T. etc., (1988), Biotechnology 6,1419-1422) pMStr59 transformed aspergillus oryzae strain BECh2 (WO 00/39322).In the 250ml bottle, cultivate transformant by the improvement FG4P substratum that the method for describing among the WO 94/26925 uses 100ml to contain 5.0AU/l Neutrase 1.5MG (Novozymes A/S, Bagsvaerd, Denmark).Neutrase is dissolved in the universal buffering liquid of pH 6.5 and filtration sterilization before interpolation.Culture was in 26 ℃, 270 rev/mins shaking culture 4 days.

Described in WO 94/26925, measure the tryptic expression of fusarium, use N-benzoyl-L-arginine-p-Nitroaniline hydrochloric acid (L-BA-pNA) as substrate, and it is as follows that the tris damping fluid of pH 8.0 comprises composition: 25mM tris, 1mM CaCl ₂With 150mM KCl.

Universal buffering liquid:

The 100mM succsinic acid

100mM HEPES

100mM CHES

100mM CABS

1mM CaCl ₂

150mM KCl

0.01% Triton X-100

Example VI-" performance test of pattern detergent washing "

In order to assess the scourability of subtilase in standard wash agent composition, can use following test conditions to carry out the standard wash experiment:

Washing composition: pattern washing composition

Detergent doses: 4.0g/l

pH： 10.1

Washing time: 20 minutes

Temperature: 30 ℃

The water hardness: 15 ° of dH

Enzyme concn: 10nm (in detergent solution)

Test macro: with the 10ml beaker of stirring rod

Yarn fabric/volume: 5 yarn fabrics

Detergent solution

Test material: EMPA 117

The component of pattern washing composition is as follows:

By adding HCl or NaOH with the pH regulator to 10.1 of detergent solution.By in test macro, adding CaCl ₂And MgCl ₂(Ca ²⁺: Mg ²⁺=4:1) water hardness is adjusted to 15 ° of dH.After washing, pieces of fabric is washed and dry air with tap water.

Use Macbeth ColorEye 7000 photometers (Macbeth, Division of KollmorgenInstruments Corporation, Germany) to measure the reflection coefficient (R of test material at 460nm _Subtilase).Method according to manufacturers is carried out measurement.

For measuring blank value, carry out the similar washing experiment of not adding enzyme.Measure reflection coefficient (R such as top just the description _Blank).

Carry out as mentioned above with reference to experiment, the scourability of wherein having tested the associated protein enzyme.Measure reflection coefficient (R such as top just the description _Savinase).

The preservation of biomaterial

Following biomaterial is preserved in Germany microbial preservation center (Mascheroder Weg 1B, D-38124Braunschweig, Germany) and given following preserving number according to the clause of budapest treaty:

The preserved material preserving number storage date

Intestinal bacteria NN049696 DSM 15940 2003-09-22

Claims

1. be selected from following proteolytic enzyme:

A. comprise the proteolytic enzyme that has the aminoacid sequence of at least 73% identity with aminoacid sequence shown in the 1-226 amino acids of SEQ ID NO:2; With

(i) complementary strand of nucleotide sequence shown in the 127-804 position Nucleotide of SEQ ID NO:1, or

(ii) subsequence of (i) of at least 100 Nucleotide; With

2. proteolytic enzyme according to claim 1, wherein said proteolytic enzyme have with aminoacid sequence shown in the 1-226 amino acids of SEQ ID NO:2 and have greater than 75.0% or greater than 80.0% or greater than 85.0% or greater than 90.0% or greater than 92.0% or greater than 94.0% or greater than 96.0% or greater than 97.0% or greater than 98.0% or greater than the aminoacid sequence of 99.0% identity.

3. proteolytic enzyme according to claim 1, it comprises the aminoacid sequence shown in the 1-226 amino acids of SEQ ID NO:2.

4. proteolytic enzyme according to claim 1, it is comprised of the aminoacid sequence shown in the 1-226 amino acids of SEQ ID NO:2.

5. according to the described proteolytic enzyme of any one in the aforementioned claim, wherein said proteolytic enzyme is the variant with proteolytic enzyme of aminoacid sequence shown in the-25-226 amino acids of SEQ ID NO:2, and described variant comprises substituting, lack and/or inserting of one or more amino-acid residues.

6. proteolytic enzyme or its variant, wherein said proteolytic enzyme is coded by the protease-encoding part that is cloned into the polynucleotide in the plasmid fragment that exists in intestinal bacteria (Escherichiacoli) DSM 15940, and the maturing part of described variant and described proteolytic enzyme has the identity greater than 73%.

7. proteolytic enzyme according to claim 6, the proteolytic enzyme maturing part that the protease-encoding of wherein said proteolytic enzyme and polynucleotide is partly encoded has greater than 75.0% or greater than 80.0% or greater than 85.0% or greater than 90.0% or greater than 92.0% or greater than 94.0% or greater than 96.0% or greater than 97.0% or greater than 98.0% or greater than 99.0% identity, wherein said polynucleotide are cloned in the plasmid fragment that exists in the intestinal bacteria of preserving number DSM 15940.

8. proteolytic enzyme according to claim 6, wherein said proteolytic enzyme comprise the proteolytic enzyme of partly being encoded by the protease-encoding that is cloned into the polynucleotide in the plasmid fragment that exists in intestinal bacteria DSM 15940.

9. proteolytic enzyme according to claim 6, wherein said proteolytic enzyme is comprised of the coded proteolytic enzyme of protease-encoding part that is cloned into the polynucleotide in the plasmid fragment that exists in intestinal bacteria DSM 15940.

10. proteolytic enzyme according to claim 1, wherein said proteolytic enzyme by under the medium stringent condition, preferably under high stringent condition with the nucleic acid sequence encoding of following sequence hybridization:

(ii) subsequence of (i) of at least 100 Nucleotide.

11. according to the described proteolytic enzyme of any one in the aforementioned claim, wherein said proteolytic enzyme is trypsin-like proteolytic enzyme.

12. according to the described proteolytic enzyme of any one in the aforementioned claim, wherein said proteolytic enzyme has at least 50% residual activity after 35 ℃ of storages, wherein determination of activity is according to " stability in the EXAMPLE V washing composition " described test.

13. proteolytic enzyme according to claim 11, wherein said proteolytic enzyme have at least 55% residual activity after 35 ℃ of storages, for example have at least 60% residual activity after 35 ℃ of storages, more preferably have at least 65% residual activity after 35 ℃ of storages.

14. the nucleotide sequence that separates, wherein said nucleotide sequence comprise the nucleotide sequence of proteolytic enzyme that any one defines in the aforementioned claim of coding.

15. institute's separated nucleic acid sequence of proteins encoded enzyme, wherein said nucleotide sequence is selected from:

A. the nucleotide sequence that has at least 80% identity with nucleotide sequence shown in the 52-804 position Nucleotide of SEQ NO:1; With

(ii) subsequence of (i) of at least 100 Nucleotide.

16. nucleotide sequence according to claim 14, wherein said nucleotide sequence have with nucleotide sequence shown in the 52-804 position Nucleotide of SEQID NO:1 and have at least 86%, such as at least 87%, for example at least 88%, preferably at least 89%, such as at least 90%, for example at least 91%, more preferably at least 92%, such as at least 93%, for example at least 94%, most preferably at least 95%, such as at least 96%, for example at least 97%, particularly at least 98%, the nucleotide sequence of preferred at least 99% identity.

17. comprise the nucleic acid construct of the described nucleotide sequence of any one among the claim 14-16, wherein said nucleic acid construct effectively is connected with one or more control sequences that can instruct proteolytic enzyme to express in suitable host.

18. recombinant expression vector, wherein said recombinant expression vector comprise nucleic acid construct, promotor and transcription termination signal and the translation termination signal of claim 17.

19. recombinant host cell, wherein said recombinant host cell comprises the nucleic acid construct of claim 17.

20. host cell according to claim 19, it is fungi or yeast, is preferably filamentous fungus, especially is Aspergillus (Aspergillus).

21. host cell according to claim 20, it is aspergillus oryzae (Aspergillus oryzae).

22. host cell according to claim 19, it is bacterium, is preferably bacillus (Bacillus), especially is bacillus lentus (Bacillus lentus).

23. for the production of the method for the described proteolytic enzyme of any one in according to claim 1-13, described method comprises:

But the defined recombinant host cell of any one in a. under the condition that the inducible protein enzyme produces, cultivating such as claim 19-22; With

B. reclaim proteolytic enzyme.

24. cleaning compositions or detergent composition, preferred laundry composition or wash the dish composition, it comprises according to claim 1 the described proteolytic enzyme of any one in-13.

25. composition according to claim 24, it additionally comprises cellulase, lipase, at, oxydo-reductase, other proteolytic enzyme, amylase or its mixture.

26. such as the purposes of proteolytic enzyme that any one defines in cleaning compositions or detergent composition among the claim 1-13.

27. the method that is used for cleaning or washs hard surface or clothing, described method comprise hard surface or clothing are contacted with defined composition among the claim 25-26.